This proposal, submitted in response to RFA-RR-05-001, describes development of an innovative methodology, rotational fluorescence correlation spectroscopy, for measurement of molecular interactions in a wide range of biomedical applications. Interactions between molecules in cellular environments are key areas of interest in contemporary cell biology. Since its introduction in 1972, fluorescence correlation spectroscopy (FCS) has proved a powerful tool for measuring translational diffusion, and thus interactions, of biological molecules. However, protein rotational motion, as regulated by the size and environment of the diffusing species, is a much more sensitive probe of such interactions than is lateral diffusion. In contrast with its wide-spread application in measuring translational diffusion, FCS has not been applied in a significant way to examination of molecular rotation. Our laboratory focuses on measurement of molecular rotation, using and developing methods for this purpose, and has contributed a substantial fraction of all work published in this area. In this project, we will explore the theoretical and experimental issues involved in implementing rotational fluorescence correlation spectroscopy and will develop of practical strategies for application to molecules in solution and in intact cells. Our specific aims are to develop a practical microscope-based system for rotational fluorescence correlation spectroscopy based on two-photon excitation (Aim 1), to develop data analysis methods for separating rotational and translational dynamics of particles from rotational FCS data (Aim 2) and to evaluate the performance of rotational FCS methods in measuring rotational dynamics of selected test systems (Aim 3). Innovative aspects of the project include new strategies for separating rotational and translational dynamics in correlation data, for extending rotational FCS measurement into the nanosecond timescale where interactions between dissolved proteins are observed and for achieving on-line analysis of rotational FCS data. Moreover, the multi-objective region detector to be constructed will, for the first time, permit true luminescence emission anisotropy to be evaluated from microscope observations without introducing a priori assumptions about motional constraints of observed molecules. [unreadable] [unreadable] [unreadable]